Project description:Differentiation of hemopoietic stem cells into granulocytes is characterized by distinct changes in the transcriptome. We analyzed mRNA expression in primary murine myeloid cells at four successive stages of hemopoietic differentiation; Lin- Sca1+ cKit+ stem/progenitor cells (LSKs), promyelocytes, myelocytes and granulocytes.
Project description:Differentiation of hemopoietic stem cells into granulocytes is characterized by distinct changes in the transcriptome. We analyzed mRNA expression in primary murine myeloid cells at four successive stages of hemopoietic differentiation; Lin- Sca1+ cKit+ stem/progenitor cells (LSKs), promyelocytes, myelocytes and granulocytes. Using fluorescence–activated cell sorting, we isolated primary murine myeloid cells at four successive stages of hemopoietic differentiation; Lin- Sca1+ cKit+ stem/progenitor cells (LSK), promyelocytes, myelocytes and granulocytes.
Project description:This study sought to determine the dynamic changes of miRNA expression during mouse granulopoiesis. We not only performed analyses of miRNA expression levels in whole cells but also analyzed purified nuclear and cytoplasmic cell fractions to profile miRNA subcellular localization. qRT-PCR analysis of miRNAs was performed on whole cell, nuclear and cytoplasmic RNAs extracted from mouse hemopoietic stem cells (LSKs), promyelocytes, myelocytes and granulocytes. 100 ng of RNA was reversed transcribed using the Taqman miRNA Reverse Transcription Kit and Megaplex RT Primers rodent pool A and B (Life Technologies). Complementary DNA (cDNA) was amplified using a TaqMan rodent microRNA A and B Array v2.0 (Life Technologies) with TaqMan Universal PCR Master Mix on an ABI 7900HT Sequence Detection System.
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). There was a significant overlap of IR between human and mouse (P=2.85E-22, hypergeometric test), showing that IR is conserved.Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression. Sequencing of polyadenylated RNA from three types of myeloid cells (promyelocytes, myelocytes and granulocytes) using Illumina GAIIx
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression. We used Affymetrix's Mouse Gene 1.0 ST array to determine accumulation of RNA-transcripts following inhibition of nonsense-mediated decay in primary mouse granulocytes using caffeine.
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression.
Project description:Using mRNA-seq, we determined intron retaining genes that were differentially regulated in FACS purified cells at three progressive stages of mouse granulopoiesis; CD34+Kit+Gr-1low promyelocytes, CD34-Kit-Gr-1mid myelocytes and CD34-Kit-Gr-1high granulocytes. We found that IR affects 86 genes, including those specific to granulocyte (Lyz2 and MMP8) and nuclear architecture (Lmnb1 and Lbr). IR was associated with the decrease in protein levels measured by mass spectrometry (P=0.0015, binomial test). There was a significant overlap of IR between human and mouse (P=2.85E-22, hypergeometric test), showing that IR is conserved.Inhibition of NMD in granulocytes resulted in marked accumulation of 39/86 intron retaining mRNAs (P<0.05, RUV procedure with Holm-Bonferroni correction), indicating that IR triggers NMD to downregulate mRNA and protein expression.
Project description:A global microRNA expression profile was obtained from gradient-purified granulocytes (>95% pure) collected at the time of screening and at cycle 4 of treatment Protocol #18424-256 is a Phase 2 study of the JAK1 and JAK2 inhibitor INCB01842 in patients with advanced polycythemia vera (PV) and essential thrombocythemia (ET) refractory to hydroxyurea; The aim was to to determine whether treatment with INC180424 was associated with changes in the global microRNA expression profile we compared granulocytes collected at baseline (screening) and at cycle 4